In 2013, the CDC estimated well over 1 million people over age 13 are living with HIV infection, with over 180,000 others infected but undiagnosed. Strict adherence to highly active/combination anti-retroviral therapy (HAART/cART) persist despite prevents full-blown AIDS, but fails to prevent complications from latent HIV reservoirs that treatment. In particular, the oral HIV reservoir is under constant reactivation due to environmental exposure to food and waterborne pathogens and a major contributing factor in HAART patient oral health problems. Indeed, the oral reservoir is an ongoing obstacle to the search for a cure to HIV-AIDS. Issues with Current Solutions & How Product Meets Unmet Needs Current methods of quantifying the latent reserve include the quantitative viral outgrowth assay (QVOA), PCR and RT-PCR, for which only PCR and RT-PCR have been applied to oral mucosa. QVOA is time and resource intensive, underestimates the reservoir. PCR greatly overestimates the latent pool by detection of unintegrated and nonfunctional HIV genomes. RT-PCR can be used to detect viral RNA to 20-50 virus particles per mL, reduces the time to result of QVOA, and is applicable for measuring viral load, but fails to directly detect replication-competent latent HIV-infected cells. Q-VOA, the accepted quantitation standard, is currently available only at relatively few AIDS research facilities, due to its intensive resource and labor requirements. This product will introduce a real-time molecular assay to detect transcriptionally-competent HIV mRNA directly from latently infected cells isolated from HAART/cART patient oral samples. With validation, this assay has the potential to provide point-of-care, real-time quantitation of the oral mucosa HIV reservoir to indicate need for and efficacy of new treatments and significantly accelerate testing and discovery of a cure for HIV infection. Summary of Approach The technology proposed is a point-of-care diagnostic based on a novel splice-site assay for multiply-spliced HIV mRNA, indicative of the replication-competent HIV proviruses in latent reservoirs. The assay is a quantitative autoligation detection reaction (qLDR) for highly sensitive and specific mRNA splice site detection. qLDR uses fluorogenic probes for chemical ligation in a standard amplification thermocycling reaction, providing real-time detection in a microfluidic device for point-of-care detection of the latent and activated HIV reservoir in the orl mucosa of HAART patients. Collaborators and Unique Resources Jan Biotech, Inc., with expertise in molecular diagnostic development, will collaborate with Associate Professor David Putnam, Department of Chemical and Biomolecular Engineering of Cornell University. Professor Harris Gelbard, investigating the phenomenon of latent reservoir neuroAIDS at the University of Rochester Center for AIDS Research (CFAR) will provide consultation and will facilitate testing of human oral mucosa samples, provided by the National Disease Research Interchange (NDRI). Cell lines will be provided by the NIH AIDS Reagent Program. The point-of-care real-time microfluidics prototype will be developed in consolation with microfluidics design engineer Scott Stelick. Phase I Specific Aims Specific Aim 1: Develop real-time spliced-RNA detection assay for quantitation of oral latent HIV-1 RNA Specific Aim 2: Test qLDR using human latent HIV-1 CD4+ cell lines and oral samples Specific Aim 3: Develop point-of-care real-time qLDR microfluidics prototype device How Anticipated Results will Justify Phase II and Further Product Development Superior performance of qLDR is expected compared to RT-PCR, with real-time, sensitive and specific detection of multiply-spliced HIV mRNA in latent HIV oral mucosa from HAART/cART patients. The prototype microfluidics device will facilitate product development to a point-of-care commercial ready clinical diagnostic. Additional Time and Funding Necessary to Bring Product to Market after Phase I Completion It is anticipated that a high-throughput laboratory research product can be brought to market as a laboratory assay kit for research purposes at the completion of the Phase II, two years after the Phase I work has been completed. It is anticipated that an additional 2-3 years and funding through a Phase II bridge award will be needed to perform the clinical trials required for FDA approval as a point-of-care clinical diagnostic.